Project Summary An enzyme like lysozyme is a major plyer in the human immune system and disease defense system. However, bacterial pathogens, in turn, have evolved resistance mechanisms to evade immune defense reaction. As consequences, super-resilient, drug-resistant pathogens are major issues in health care. The long-term objective of this proposal is to offer insight into the molecular interaction of different ligands including modified-substrates and inhibitors to lysozyme, which is a prerequisite for the development of novel antibacterial agents. Three integrated specific aims are proposed to reveal a precise molecular mechanism that controls dynamic protein-ligand interaction using a novel, electronic-type single-molecule approach developed by the PI. This approach interrogates enzyme dynamics and its involvement in ligand binding, catalysis, and inhibition at unprecedented time scales over extended periods of time measurements. With the invention, we will initially investigate the enzyme fluctuation and flexibility to determine their contribution to the ligand recognition and binding, as well as their roles to the enzyme catalysis and inhibition. Next, we will dissect the structural and molecular basis of enzyme-ligand interaction with various mimic antibacterial inhibitors, including modified-substrates and peptide-inhibitors. In addition, we plan to drive the fluctuation and examine the effects on enzyme activity. The research attempts to understand whether the driven-fluctuation can interfere with, or even control, enzyme activity and function, either by inhibiting or amplifying the native activity. The outcome of the proposed research will help establish a structural basis for the design of compounds that can suppress super-resilient, drug-resistant pathogens as well as provide new possibilities for diagnostic devices that bridge electronics and biochemical function.